The prospect of employing antisense nucleic acids (ASNA) for treating human malignancies remains tantalizing, but unrealized. We hypothesize that ASNA would make better drugs if fundamental problems related to mRNA target sequence selection, and molecule delivery, were solved. The goal of this project is to address these core issues in the following specific aims: Aim #1- Develop A Rational Method For Targeting ASNA- We have developed self-quenching reporter molecules (SQRM) that signal only after hybridization, and have used these probes to "map" hybridization accessible sites in mRNA. We will determine the in vivo utility of this strategy by mapping additional target mRNAs, and measuring the efficiency with which sequence directed ASNA inhibit gene expression in living cells. The hypothesis that naturally occurring intracellular proteins might enhance ASNA/mRNA hybridization will also be tested. Candidate proteins will be identified by function, and by using affinity chromatography, and mass spectrometry. Finally, the utility of DNA backbones with enhanced strand invasion properties, with or without proteins that facilitate hybridization, will also be explored; Aim #2- Test the Hypothesis that Gene Silencing Efficiency of Rationally Targeted ASNA Can be Enhanced by Backbone, Sequence, or Pendant Modifications- ASNA cleave mRNA by enzymatic activity engineered into the molecule, or by activating endogenous RNaseH. We will examine the ability of rationally targeted ASNA, synthesized with various backbone modifications, to cleave mRNA targets in vitro and in vivo. The ability of SQRM with pendants that can be photoactivated to detect, and kill, cells on the basis of target mRNA expression will also be examined; Aim #3. Examine the ability of rationally designed, activity optimized ASNA to detect, and kill, tumor cells in animal models of human leukemia- Pharmacodynamic studies of optimized molecules will be undertaken, and their ability to serve as diagnostic, and therapeutic molecules in animal models of human leukemia will be explored.